Sensitivity of Four Various Candida Species to Photodynamic Therapy Mediated by Indocyanine Green, an in vitro Study

Statement of the Problem: Various species of candida contribute to oral candidiasis. It is the time to shift from conventional rigid antimicrobial therapies to more patient specific and safer ones. Purpose: The present study aimed to investigate antifungal effects of photodynamic therapy (PDT) using Indocyanine green as photosensitizer and low-power laser irradiation on the viability of candida albicans, candida tropicalis, candida glabrata and candida krusei, and to compare it with Nystatin as the conventional treatment. Materials and Method: In this in vitro study, 0.5 McFarland suspensions of candida's species were prepared (n=50, each). Each strain was then divided into five groups of 10 samples each, according to the following experimental interventions: (1) Nystatin, (2) photodynamic therapy: laser irradiation (wavelength= 808 nm, power= 100 mW, energy density= 10 J/cm2, exposure duration= 100 s) in the presence of the photosensitizer, (3) laser irradiation alone, (4) treatment with the PS alone and (5-control: no exposure to laser light or photosensitizer. Next, serial dilutions were prepared and seeded onto Sabouraud dextrose agar. The colonies were counted, and the values of log (CFU/ml) were analyzed by variance and the Tamhan test (p< 0.05). Results: Photodynamic therapy mediated indocyanine green is significantly effective in reducing the number of CFU/ml of all species of candida tested, compared to control group (p< .001). Nystatin, laser irradiation and photodynamic therapy, with respectively decreasing potency, have considerably reduced the number of candida's colonies in all four bacterial strains (p<. 05). Photosensitizer alone, showed impressive antimicrobial potency against all species of candida except candida albicans, in comparison to control group. Conclusion: It seems that laser therapy alone is more powerful than photodynamic therapy mediated indocyanine green; however, conventional treatment has still the top antimicrobial efficacy towards all candida species.


Introduction
Oral candidiasis is an opportunistic infection in the oral cavity. Candida albicans (C.albicans) is the most prevalent cause of oral mucosa candidiasis, which reaches to 60-70 percent of cases [1]. C.albicans the form of "nonpathogen," role as the normal flora of mouth, though under special conditions, it may lead to candidiasis [2].
Today, there are various antifungal medications introduced in this regard such as ionophores (Nystatin and Amphotericin B) and azole drugs. However, there are still some limitations in facile prescription of these drugs such as the bitter taste of Nystatin, which leads to nausea and patient intolerance [3], or the occurrence of drug resistance, especially to azoles, as was described in 81 percent of patients with HIV infection, who were under oral C.albicans therapy [4]. Moreover, oral candidiasis may occur iatrogenic as an adverse effect of other treatments such as chemotherapy for cancer suppression; in these patients, drug resistance was also abundantly observed [5]. These findings and coinfection with microbial flora such as Pseudomonas Aeruginosa necessitate a powerful, universal attempt to develop a novel strategy to remove fungal infections, without causing harmful effects or inducing resistance reactions [6][7][8].
Photodynamic therapy (PDT) assisted laser is a new modality, based on non-toxic photosensitizer (PS) and safe light source. The combination of these items can trigger a biological cascade in the presence of radical oxygen for apoptosis and annihilation of microorganisms as long as malignant cells [9]. Different PSs (such as methylene blue, indocyanine green and toluidine blue) as well as diverse laser parameters were examined in this regard to achieve the best result in elimination of microbes and malignant cells. The indocyanine green as a new PS which followed by laser illumination, has shown promising effects against periodontal and periimplant pathogens and malignant cells of melanoma [10][11][12]. The present study was to compare the susceptibility of four various species of candida to PDT, PS, laser irradiation, and to conventional treatment of Nystatin. in each group were selected randomly as control (N= 10), which did not receive any treatment or intervention.

Microorganisms and culture conditions
Freeze-dried candida species have been subjected to revive the process. In order to prepare.  Table 1.   Figure 1).

Data analysis
The data have been statistically analyzed by SPSS23 software One-way ANOVA test and Tamhan test have been used to detect any significant difference between groups (p< 0.05).

Results
C.albicans counts (CFU/ml) were transformed into base -10 logarithms. Therefore, one-way ANOVA with followed Tamhan test for multiple comparisons was performed ( Table 2). The results of dual comparison of the  Table 3.
All interventions showed anti-fungal efficacy when compared to control, although species of C.albicans did not reduce effectively in comparison to control group when subjected to PS alone. In total, the highest number of candida's colonies was seen in a control group, while the least was achieved in Nystatin group.
C.glabrata, which showed the most sensitivity to Nystatin, was eliminated. C.krusei showed the least sensitivity to Nystatin. Additionally, this trend was also found with PDT employment. C.tropicalis was recognized as the most sensitive strain to laser irradiation; inversely, C.glabrata was the most resistant to this.
Surprisingly, PS only, also showed anti-fungal efficacy with the most promising effects on C.tropicalis and the least on C.glabrata strain.
Laser irradiation was significantly more effective than PDT against C.krusei and C.tropicalis; however, when the laser employed on C.glabrata and C.albicans strains, it showed a statistically equal anti-fungal power.
PDT reduced candida colonies more effective than PS alone except in samples of C.albicans, which showed no statistically significant difference (p= 0.13).   In an in vitro study performed by Fekrazad et al. [13], the effects of PDT mediated indocyanine green and new methylene blue against C.albicans was compared with control (no treatment). Similar to the results of present study, they reported a promising anti-fungal effect of PDT [13].

Present investigation evaluated anti-fungal efficacy of
Azizi et al. [14], also investigated into the in vitro e-  [13][14]. This conclusion demonstrated that the laser illumination following PS application in PDT is of paramount importance; therefore, it is suggested to make forthcoming investigations with a focus more on selecting the best laser parameters' layout than the type or characteristics of PS.
The characteristics of the aimed microorganism have a key role in PDT success rate. C.albicans seems to be more resistant than gram-positive bacteria against this treatment modality. It is suggested that perhaps the presence of nuclear membrane, larger cell size, and the fewer target areas for free oxygen radicals per unit of cell volume in C.albicans may play a significant role in this resistance [20]. In the present study as well, C.albicans was more resistant to PDT compared to other employed species of candida.

Maximum absorption of light by colored PS mole-
cules is also an important issue when applying PDT.
The wavelength of radiation must be set where there is the most absorbance by the PS molecules. This will produce a maximum amount of oxygen free radical to eliminate the target microorganism. For example, the most absorbent of the indocyanine green is in the range of 805-810 nm [21][22]. Therefore, it seems that the wavelength used in the present study was optimum; however, the authors believe that higher-energy density of the laser was needed to achieve better results. Unfortunately, in previously published studies, different laser parameters with different conditions and methods have been examined, which make the comparison and consensus between various and contradictory results very difficult [14,23]. In the present study laser, the parameters were chosen according to Azizi et al. [14] investigation.
One of the critical concerns about laser application on vital tissues is the laser side effect in increasing the targeted tissue temperature, shifting the tissue healing pathways to unwanted and harmful ones. Nonetheless, it is demonstrated that diode lasers in comparison to other laser types would produce less heat [24]. Silva et al. [25] reported an increase of 2 centigrade in temperature of target tissue when applying PDT (by diode laser) for 30, 60, and 120 seconds, which can be negligible. In another study conducted by Hirata et al. [26], laser application in power ranging 50-500mW for 2min duration caused moderator effects on mammalian cell proliferation in vitro [26]. It seems that these side effects could be less prominent when time duration of laser illumination is reduced. The toxicity of indocyanine green has not been demonstrated, up to date [25].
Wainwright et al. [27] demonstrated that microor- Previous data confirmed PDT usability in dental practice for anti-microbial purposes [28][29]. The effects of PDT induced by indocyanine green have been demonstrated as an effective treatment modality for periodontal disease [11][12] and also for eradication of melanomas and acne vulgaris [10,30]. Nevertheless, there are still some unknown points, which prevent it to be as a standard modality; it seems that in vitro studies are yet needed to standardize various options and parameters introduced in literature. Furthermore, comparative investigations with conventional therapies should be held to assess the cost benefit of this novel modality.
In the present study, Nystatin showed significant better results than PDT, so the authors of this study propose anti-candida PDT application when Nystatin therapy is impossible or tolerated or as a conjunctive therapy to traditional treatments.

Indocyanine green is approved by Food and Drug
Administration (FDA) and has been used for diagnostic purposes in the field of medicine, such as detection of capillary roots [31]. Absorption wavelength of indocyanine green is at 805 nm. It is demonstrated that indocyanine green binds to plasma proteins, so does not lead to chemical changes in the body [11].
In the present study, the efficacy of laser with or without PS, application was nearly equal, while Fekrazad et al.'s [13] investigation of laser illumination following indocyanine green application showed considerable better anti-fungal results than laser alone. This may be due to different brands of indocyanine green used in the two investigations, as we used Cardiogreen and they employed Emundo (both by Sigma Aldrich Company).
Accordingly, it seems that Emundo has more potency in this field of application than Cardiogreen. Nonetheless, this hypothesis should be examined more specifically, in a matched comparative study. Moreover, laser parameters and mode of PS application as well as the method of PS dilution were different in these two investigations; each one could have a critical role in achieving dissimilar results.

Conclusion
Under the conditions of the present study, susceptibility of all species of candida was primarily to Nystatin. PDT is significantly effective in reducing the candida colonies. PS alone, showed sufficient antimicrobial potency against all species of candida except C.albicans, in comparison to control group. The outlook for new treatments in candidiasis seems to be very clear. Further studies on the efficacy of these treatments are recommended.